Hyaluronic acid hydrogels incorporating platelet lysate enhance human pulp cell proliferation and differentiation

  • Leopoldina D. F. Almeida
  • Pedro S. Babo
  • Cristiana R. Silva
  • Márcia T. Rodrigues
  • Josimeri Hebling
  • Rui L. Reis
  • Manuela E. Gomes
Special Issue: ESB 2017 Original Research
Part of the following topical collections:
  1. Special Issue: ESB 2017


The restoration of dentine-pulp complex remains a challenge for dentists; nonetheless, it has been poorly addressed. An ideal system should modulate the host response, as well as enable the recruitment, proliferation and differentiation of relevant progenitor cells. Herein was proposed a photocrosslinkable hydrogel system based on hyaluronic acid (HA) and platelet lysate (PL). PL is a cocktail of growth factors (GFs) and cytokines involved in wound healing orchestration, obtained by the cryogenic processing of platelet concentrates, and was expected to provide the HA hydrogels specific biochemical cues to enhance pulp cells’ recruitment, proliferation and differentiation. Stable HA hydrogels incorporating PL (HAPL) were prepared after photocrosslinking of methacrylated HA (Met-HA) previously dissolved in PL, triggered by the Ultra Violet activated photoinitiator Irgacure 2959. Both the HAPL and plain HA hydrogels were shown to be able to recruit cells from a cell monolayer of human dental pulp stem cells (hDPSCs) isolated from permanent teeth. The hDPCs were also seeded directly over the hydrogels (5 × 104 cells/hydrogel) and cultured in osteogenic conditions. Cell metabolism and DNA quantification were higher, in all time-points, for PL supplemented hydrogels (p < 0,05). Alkaline phosphatase (ALPL) activity and calcium quantification peaks were observed for the HAPL group at 21 days (p < 0,05). The gene expression for ALPL and COLIA1 was up-regulated at 21 days to HAPL, compared with HA group (p < 0,05). Within the same time point, the gene expression for RUNX2 did not differ between the groups. Overall, data demonstrated that the HA hydrogels incorporating PL increased the cellular metabolism and stimulate the mineralized matrix deposition by hDPSCs, providing clear evidence of the potential of the proposed system for the repair of damaged pulp/dentin tissue and endodontics regeneration.



LFDA acknowledges Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for the grant 2014/12017-8. Portuguese Foundation for Science and Technology (FCT) for PSB PhD grant SFRH/BD/73403/2010, MTR post-doctoral grant (SFRH/BPD/111729/2015), MEG grant (IF/00685/2012), and RECOGNIZE project (UTAP-ICDT/CTM-BIO/0023/2014), RL3-TECT - NORTE-07-0124-FEDER-000020 project co-financed by ON.2 (NSRF) through ERD. This study also received financial support from FCT/Ministério da Ciência, Tecnologia, e Ensino Superior (FCT/MCTES) and Fundo Social Europeu through Programa Operacional do Capital Humano (FSE/POCH) PD/59/2013 for the LA ICVS-3Bs (UID/Multi/50026/2013). The authors would like to thank Maurizio Gulino, for its support in the in vitro experiments and Maló Clinic, Porto, Dra Ana Ferro and Dr Bruno Queridinha for the donation of permanent teeth.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Silva CR, Gomez-Florit M, Babo PS, Reis RL, Gomes ME. 3D Functional scaffolds for dental tissue engineering. Funct. 3D Tissue Eng. Scaffolds. Elsevier; 2018. pp. 423–50.Google Scholar
  2. 2.
    Ferroni L, Gardin C, Sivolella S, Brunello G, Berengo M, Piattelli A, et al. A hyaluronan-based scaffold for the in vitro construction of dental pulp-like tissue. Int J Mol Sci. 2015;16:4666–81.CrossRefGoogle Scholar
  3. 3.
    Chrepa V, Austah O, Diogenes A. Evaluation of a commercially available hyaluronic acid hydrogel (restylane) as injectable scaffold for dental pulp regeneration: an in vitro evaluation. J Endod. 2017;43:257–62.CrossRefGoogle Scholar
  4. 4.
    Inuyama Y, Kitamura C, Nishihara T, Morotomi T, Nagayoshi M, Tabata Y, et al. Effects of hyaluronic acid sponge as a scaffold on odontoblastic cell line and amputated dental pulp. J Biomed Mater Res Part B Appl Biomater. 2010;92B:120–8.CrossRefGoogle Scholar
  5. 5.
    Soares DG, Rosseto HL, Basso FG, Scheffel DS, Hebling J, Costa CA, de S. Chitosan-collagen biomembrane embedded with calcium-aluminate enhances dentinogenic potential of pulp cells. Braz Oral Res. 2016;30:1–10.Google Scholar
  6. 6.
    Dobie K, Smith G, Sloan AJ, Smith AJ. Effects of alginate hydrogels and TGF-β1 on human dental pulp repair in vitro. Connect Tissue Res. 2002;43:387–90.CrossRefGoogle Scholar
  7. 7.
    Kitamura C, Nishihara T,Terashita M,Tabata Y,Washio A. Local regeneration of dentin-pulp complex using controlled release of FGF-2 and naturally derived sponge-like scaffolds. Int J Dent. 2012;2012:1–8.CrossRefGoogle Scholar
  8. 8.
    Ishimatsu H, Kitamura C, Morotomi T, Tabata Y, Nishihara T, Chen KK, et al. Formation of dentinal bridge on surface of regenerated dental pulp in dentin defects by controlled release of fibroblast growth factor-2 from gelatin hydrogels. J Endod. 2009;35:858–65.CrossRefGoogle Scholar
  9. 9.
    Qu T, Jing J, Ren Y, Ma C, Feng JQ, Yu Q, et al. Complete pulpodentin complex regeneration by modulating the stiffness of biomimetic matrix. Acta Biomater. 2015;16:60–70.CrossRefGoogle Scholar
  10. 10.
    Piva E, Silva AF, Nör JE. Functionalized scaffolds to control dental pulp stem cell fate. J Endod. 2014;40:S33–40.CrossRefGoogle Scholar
  11. 11.
    Suzuki T, Lee CH, Chen M, Zhao W, Fu SY, Qi JJ, et al. Induced migration of dental pulp stem cells for in vivo pulp regeneration. J Dent Res. 2011;90:1013–8.CrossRefGoogle Scholar
  12. 12.
    Galler KM, Cavender AC, Koeklue U, Suggs LJ, Schmalz G, D’Souza RN. Bioengineering of dental stem cells in a PEGylated fibrin gel. Regen Med. 2011;6:191–200.CrossRefGoogle Scholar
  13. 13.
    Yang JW, Zhang YF, Sun ZY, Song GT, Chen Z. Dental pulp tissue engineering with bFGF-incorporated silk fibroin scaffolds. J Biomater Appl. 2015;30:221–9.CrossRefGoogle Scholar
  14. 14.
    Jih G,Wu H,Chen M, Chen M, Chang S, Wang T. Control of three-dimensional substrate stiffness to manipulate mesenchymal stem cell fate toward neuronal or glial lineages. Acta Biomaterialia. 2013;9:5170–80.CrossRefGoogle Scholar
  15. 15.
    Shibata S, Yoneda S, Yanagishita M, Yamashita Y. Isolation of proteoglycan (versican) aggregate from rat dental pulp. Arch Oral Biol. 2000;45:563–8.CrossRefGoogle Scholar
  16. 16.
    Babo PS, Reis RL, Gomes ME. Production and characterization of hyaluronic acid microparticles for the controlled delivery of growth factors using a spray/dehydration method. J Biomater Appl. 2016;31:693–707.CrossRefGoogle Scholar
  17. 17.
    Domingues RMA, Silva M, Gershovich P, Betta S, Babo P, Caridade SG, et al. Development of injectable hyaluronic acid/cellulose nanocrystals bionanocomposite hydrogels for tissue engineering applications. Bioconjug Chem. 2015;26:1571–81.CrossRefGoogle Scholar
  18. 18.
    Babo PS, Pires RL, Santos L, Franco A, Rodrigues F, Leonor I, et al. Platelet lysate-loaded photocrosslinkable hyaluronic acid hydrogels for periodontal endogenous regenerative technology. ACS Biomater Sci Eng. 2017;3:1359–69.CrossRefGoogle Scholar
  19. 19.
    Neves LS, Babo PS, Gonalves AI, Costa-Almeida R, Caridade SG, Mano JF, et al. Injectable hyaluronic acid hydrogels enriched with platelet lysate as a cryostable off-the-shelf system for cell-based therapies. Regen Eng Transl Med. 2017;3:53–69. Scholar
  20. 20.
    Burdick JA, Chung C, Jia X, Randolph MA, Langer R. Controlled degradation and mechanical behavior of photopolymerized hyaluronic acid networks. Biomacromolecules. 2005;6:386–91.CrossRefGoogle Scholar
  21. 21.
    Fekete N, Gadelorge M, Fürst D, Maurer C, Dausend J, Fleury-Cappellesso S, et al. Platelet lysate from whole blood-derived pooled platelet concentrates and apheresis-derived platelet concentrates for the isolation and expansion of human bone marrow mesenchymal stromal cells: production process, content and identification of active comp. Cytotherapy. 2012;14:540–54.CrossRefGoogle Scholar
  22. 22.
    Crespo-Diaz R, Behfar A, Butler GW, Padley DJ, Sarr MG, Bartunek J, et al. Platelet lysate consisting of a natural repair proteome supports human mesenchymal stem cell proliferation and chromosomal stability. Cell Transplant. 2011;20:797–812.CrossRefGoogle Scholar
  23. 23.
    Zimmermann R, Jakubietz R, Jakubietz M, Strasser E, Schlegel A, Wiltfang J, et al. Different preparation methods to obtain platelet components as a source of growth factors for local application. Transfus. 2001;41:1217–24.CrossRefGoogle Scholar
  24. 24.
    Tziafas D. Basic mechanisms of cytodifferentiation and dentinogenesis during dental pulp repair. Int J Dev Biol. 2017;39:281–90.Google Scholar
  25. 25.
    Chase LG, Lakshmipathy U, Solchaga LA, Rao MS, Vemuri MC. A novel serum-free medium for the expansion of human mesenchymal stem cells. Stem Cell Res Ther. 2010;1:8.CrossRefGoogle Scholar
  26. 26.
    Costa-Almeida R, Franco AR, Pesqueira T, Oliveira MB, Babo PS, Leonor IB, Mano JF, Reis RL, Gomes ME. The effects of platelet lysate patches on the activity of tendon-derived cells. Acta Biomaterialia. 2018;68:29–40.CrossRefGoogle Scholar
  27. 27.
    Nör JE. Buonocore memorial lecture. Oper Dent. 2006;31:633–42.CrossRefGoogle Scholar
  28. 28.
    Silva ED, Babo PS, Costa-Almeida R, Domingues RMA, Mendes BB, Paz E, et al. Multifunctional magnetic-responsive hydrogels to engineer tendon-to-bone interface. Nanomed Nanotechnol Biol Med. 2017.Google Scholar
  29. 29.
    Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and invivo. Proc Natl Acad Sci. 2000;97:13625–30.CrossRefGoogle Scholar
  30. 30.
    Galler KM, Buchalla W, Hiller KA, Federlin M, Eidt A, Schiefersteiner M, et al. Influence of root canal disinfectants on growth factor release from dentin. J Endod. 2015;41:363–8.CrossRefGoogle Scholar
  31. 31.
    Mao JJ, Kim SG, Zhou J, Ye L, Cho S, Suzuki T, et al. Regenerative endodontics. Dent Clin North Am. 2012;56:639–49.CrossRefGoogle Scholar
  32. 32.
    Woo S, Kim W, Lim H. Combination of mineral trioxide aggregate and platelet-rich fibrin promotes the odontoblastic differentiation and mineralization of human dental pulp cells via BMP/Smad signaling pathway. Journal of Endodontics. 2016;42:82–8.CrossRefGoogle Scholar
  33. 33.
    Fortunato TM, Beltrami C, Emanueli C, De Bank PA, Pula G. Platelet lysate gel and e ndothelial progenitors stimulate microvascular network formation in vitro: tissue engineering implications. Sci Rep. 2016;6:25326.Google Scholar
  34. 34.
    Banchs F, Trope M. Revascularization of immature permanent teeth with apical periodontitis: new treatment protocol? J Endod. 2004;30:196–200.CrossRefGoogle Scholar
  35. 35.
    Shivashankar VY, Johns DA, Vidyanath S, Kumar MrR, Shivashankar VY. Platelet rich fibrin in the revitalization of tooth with necrotic pulp and open apex. J Conserv Dent. 2012;15:395.CrossRefGoogle Scholar
  36. 36.
    Chen B, Sun H-H, Wang H-G, Kong H, Chen F-M, Yu Q. The effects of human platelet lysate on dental pulp stem cells derived from impacted human third molars. Biomaterials. 2012;33:5023–35.CrossRefGoogle Scholar
  37. 37.
    Apel K, Hirt H. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol. 2004;55:373–99.CrossRefGoogle Scholar
  38. 38.
    Wu R, Yu Y, Yin Y, Zhang X-Y, Gao L, Chen F-M. Platelet lysate supports the in vitro expansion of human periodontal ligament stem cells for cytotherapeutic use. J Tissue Eng Regen Med. 2017;11:2261–75.CrossRefGoogle Scholar
  39. 39.
    Santo VE, Babo P, Amador M, Correia C, Cunha B, Coutinho DF, et al. Engineering enriched microenvironments with gradients of platelet lysate in hydrogel fibers. Biomacromolecules. 2016;17:1985–97.CrossRefGoogle Scholar
  40. 40.
    Altaie A. Use of platelet lysate for bone regeneration-are we ready for clinical translation? World J Stem Cells. 2016;8:47.CrossRefGoogle Scholar
  41. 41.
    Copland IB, Garcia MA, Waller EK, Roback JD, Galipeau J. The effect of platelet lysate fibrinogen on the functionality of MSCs in immunotherapy. Biomaterials. 2013;34:7840–50.CrossRefGoogle Scholar
  42. 42.
    Renn T-Y, Kao Y-H, Wang C-C, Burnouf T. Anti-inflammatory effects of platelet biomaterials in a macrophage cellular model. Vox Sang. 2015;109:138–47.CrossRefGoogle Scholar
  43. 43.
    Snyder TN, Madhavan K, Intrator M, Dregalla RC, Park D. A fibrin/hyaluronic acid hydrogel for the delivery of mesenchymal stem cells and potential for articular cartilage repair. J Biol Eng. 2014;8:10.CrossRefGoogle Scholar
  44. 44.
    Zhang Y, Heher P, Hilborn J, Redl H, Ossipov DA. Hyaluronic acid-fibrin interpenetrating double network hydrogel prepared in situ by orthogonal disulfide cross-linking reaction for biomedical applications. Acta Biomater. 2016;38:23–32.CrossRefGoogle Scholar
  45. 45.
    Golub EE, Boesze-Battaglia K. The role of alkaline phosphatase in mineralization. Curr Opin Orthop. 2007;18:444–8.CrossRefGoogle Scholar
  46. 46.
    Gaur T, Lengner CJ, Hovhannisyan H, Bhat RA, Bodine PVN, Komm BS, et al. Canonical WNT signaling promotes osteogenesis by directly stimulating Runx2 gene expression. J Biol Chem. 2005;280:33132–40.CrossRefGoogle Scholar
  47. 47.
    Shui C, Spelsberg TC, Riggs BL, Khosla S. Changes in Runx2/Cbfa1 expression and activity during osteoblastic differentiation of human bone marrow stromal cells. J Bone Miner Res. 2003;18:213–21.CrossRefGoogle Scholar
  48. 48.
    Xiao G, Jiang D, Gopalakrishnan R, Franceschi RT. Fibroblast growth factor 2 induction of the osteocalcin gene requires MAPK activity and phosphorylation of the osteoblast transcription factor, Cbfa1/Runx2. J Biol Chem. 2002;277:36181–7.CrossRefGoogle Scholar
  49. 49.
    Kubota K, Sakikawa C, Katsumata M, Nakamura T, Wakabayashi K. Platelet-derived growth factor BB secreted from osteoclasts acts as an osteoblastogenesis inhibitory factor. J Bone Miner Res. 2002;17:257–65.CrossRefGoogle Scholar
  50. 50.
    Malhotra A, Pelletier MH, Yu Y, Walsh WR. Can platelet-rich plasma (PRP) improve bone healing? A comparison between the theory and experimental outcomes. Arch Orthop Trauma Surg. 2013;133:153–65.CrossRefGoogle Scholar
  51. 51.
    Oryan A, Alidadi S, Moshiri A. Platelet-rich plasma for bone healing and regeneration. Expert Opin Biol Ther. 2016;16:213–32.CrossRefGoogle Scholar
  52. 52.
    Oliveira SM, Santo VE, Gomes ME, Reis RL, Mano JF. Layer-by-layer assembled cell instructive nanocoatings containing platelet lysate. Biomaterials. 2015;48:56–65.CrossRefGoogle Scholar
  53. 53.
    Sasaki T, Kawamata-Kido H. Providing an environment for reparative dentine induction in amputated rat molar pulp by high molecular-weight hyaluronic acid. Arch Oral Biol. 1995;40:209–19.CrossRefGoogle Scholar
  54. 54.
    Leotot J, Coquelin L, Bodivit G, Bierling P, Hernigou P, Rouard H, et al. Platelet lysate coating on scaffolds directly and indirectly enhances cell migration, improving bone and blood vessel formation. Acta Biomater. 2013;9:6630–40.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Leopoldina D. F. Almeida
    • 1
    • 2
    • 3
    • 4
  • Pedro S. Babo
    • 3
    • 4
  • Cristiana R. Silva
    • 3
    • 4
  • Márcia T. Rodrigues
    • 3
    • 4
  • Josimeri Hebling
    • 2
  • Rui L. Reis
    • 3
    • 4
    • 5
  • Manuela E. Gomes
    • 3
    • 4
    • 5
  1. 1.Department of Clinical and Social DentistryFederal University of ParaíbaJoão PessoaBrazil
  2. 2.Department of Orthodontics and Pediatric Dentistry, Araraquara Dental SchoolState of São Paulo UniversityAraraquaraBrazil
  3. 3.3B’s Research Group, I3Bs–Research Institute on Biomaterials, Biodegradables and BiomimeticsUniversity of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Ciência e Tecnologia, Zona Industrial da GandraGuimarãesPortugal
  4. 4.ICVS/3B’s-PT Government Associate LaboratoryBraga/GuimarãesPortugal
  5. 5.The Discoveries Centre for Regenerative and Precision MedicineHeadquarters at University of MinhoGuimarãesPortugal

Personalised recommendations